1. Field of the Invention
The present invention relates to a method for measuring and calibrating sample injection volume and mobile phase delivery rate in a ultra micro-scale liquid phase delivery system, particularly a method using radiochemical substances and radiochemical analytical techniques to make the range of measurement or calibration achieve micro-volume or micro-flow rate grades.
2. Description of the Prior Art
Ultra micro-scale liquid phase delivery technology is the main trend and the key technology of development and application in the biomedicine, clinical diagnosis, drug screening, nano science and technology, and analytical technique currently and in the future, with its scope covering, for example, micro-liquid chromatography (μ-LC), capillary liquid chromatography, liquid chromatography mass spectrometer (LCMS), microdialysis (MD), lab-on-a-chip or microfluidic biochip, microarray biochip, micro total analysis system (μ-TAS), micro flow injection analysis (μ-FIA) or chip arrayer etc. Components such as μ-LC pump, capillary LC pump, syringe pump, micro driven spray and dipper must be used as delivery tools for samples and mobile phases.
As for the current, more mature ultra micro-scale liquid phase delivery technology, although the effective lower limit of these delivery tools for mobile phase delivery may be about 0.1 μL/min (for example, capillary LC pump) or 0.1 μL/h (for example, syringe pump), and the effective lower limit for sample single injection or spray volume is about 0.01 μL (for example, capillary LC syringe) or 0.1 nL (for example, chip arrayer), there is no convenient, rapid technology or apparatus for the measurement and calibration of mobile phase delivery rate and sample injection or spray volume.
(1) Mobile Phase Delivery Rate
The technologies known for measuring the flow rate of a fluid system include mechanical turbine, pressure difference (pneumotachometer), thermal sensitive, electromagnetic or ultrasonic technologies etc., with a measuring range from L/s to mL/min. However, the measuring methods mentioned above are not applicable for measuring the flow rate in an ultra micro-scale fluid system. For example, in Jian-zhong Fu et al., “the Architecture of a Novel Thermal Pulse Microflowmeter” (Patent No. TW 384,392), the fundamental principle is to allow the fluid to be measured flow through a micro channel, and to set a heater in the upstream of the micro channel for heating, then to set several thermal sensor modules in the downstream of the micro channel to sense the heated fluid, then to calculate the time difference and then to make it divided by the product of cross-sectional area of the micro channel and the distance between the two points. Although the principle of the invention is quite simple, the difficultly lies in that if the resolution of the flow rate measurement needs to be increased, the measured time difference will be increased or the distance between the thermal sensor modules will be decreased. Therefore, the measured time difference and the layout of thermal sensor modules in the invention will be confined by space. In addition, according to the data disclosed in the invention by Jian-zhong Fu et al., the flow range will drop from 5475 sccm to 608 sccm when the measuring time ranges from 0.1 mS to 0.9 mS. The reference also discloses that accurate time control, including suitable reaction time of temperature transmission (heat balance time) being required to be less than 0.1 mS, is a key technology which is difficult to meet by using the layout.
“Pneumotachometer” invented by Ying-song Xu (Patent No. TW 483,526), which contains a torpedo sinker type sinking flow meter having induction components which may convert pressure differences between the front-end pitot hole (over against the flowage direction of the fluid) and the static vents (multiple surrounding the surface of the torpedo sinker, in symmetrical distribution) to electronic signal, is put into a moving fluid by a sling to measure the flow rate of the fluid. The difficulty lies in that the bulk of the design is too large, and thus is not suitable for the operation of being placed directly into a micro channel which has an inner diameter between several mm to several μm.
“A Method for Setting Flow Coefficients and the Flow Measuring apparatus Using the method” (Patent No. TW 407,197) invented by Kouji Kennsan provides mainly a method for setting flow coefficients by establishing the functional relation between the flow coefficients and the flow rate. It also discloses the concept of an apparatus which may be used for setting flow coefficients and measuring the flow rate of a thermal flow sensor and an ultrasonic flowmeter in the patent. However, according to the functional relation between flow coefficients and flow rates disclosed in the invention by Kouji Kennsan (as shown in FIGS. 4-7 and FIGS. 10-19 of the specification), the ranges of flow rates are all up to meter/second with sensor parts being relatively larger. Therefore, the design is not applicable for an ultra micro-scale flow system.
In “Ultrasonic Flowmeter” (Patent No. TW 523,580) invented by Imaigu and Takataaki, the flow rate of a fluid is measured by the differences between the ultrasonic transmission times when the liquid flows through the two measurement units on a measuring tube. However, the difficulty of the design lies in that the measuring process is easily disturbed by perturbation, thereby increasing the uncertainty. In addition, factors such as large parts also make the design inapplicable for an ultra micro-scale flow system.
As compared with the known methods mentioned above, an optical fluid flowmeter system is relatively applicable for measuring the flow rate of a micro-scale system. Common fluid measurement methods include particle image velocimetry (PIV) and laser doppler velocimetry (LDV). PIV is a technology for measuring flow rates by an optical method. In experiments, some small particles are added into a flowing medium and the distances between the small particles are recorded by a secondary photography and then divided by the time intervals of the secondary photography to calculate the flow rate of the medium. The advantages of PIV include simple principle, easy-to-process data, high accuracy and measuring range up to below μL/min. However, its disadvantages include the uncertainty of measurement due to the differences between the flow rate at the center and that of the tube wall when the fluid moves in the micro channel in the mode of laminar flow, and a measurement deviation that is difficult to be calibrated when perturbation and eddy of flow arise in the micro channel. The measurement uncertainty and deviation will increase with the decrease of the tube diameter of the micro channel and the increase of the flow rates. In addition, PIV technology for measuring the micro diameter scope is known as μPIV, wherein a pulse type Nd: YAG laser light source must be used to create enough rapid and high brightness exposure to avoid blurring caused by rapid moving particles. However, the disadvantage of the method is that a microscope digital photographic equipment must be used. Therefore, there are difficulties in microscope focusing, injection and control of micron particles in experiment. A complicated and expensive pulse type equipment of Nd: YAG laser light source is also one of the deficiencies. The principle of LDV is that the wavelength of a reflected light is measured after the delivery of a monochromatic light laser wave. According to the Doppler principle, the wavelength variance of reflected lights is a function of relative moving rate of an object. Therefore, the moving rate of the object can be calculated by the wavelength variance of reflected lights. Although the measuring scope of flow rates by LDV may be between mL/min and μL/min, the disadvantage lies in that equipments of monochromatic light Helium-Neon laser or argon ion laser system used in experiments are significantly complicated and expensive. Furthermore, the uncertainty and deviation in laser wavelength measurement may be increased due to absorption and dispersion by the fluid medium. Occasionally, a high reflective efficiency substance, such as a small particle reflecting bead, must be added to increase the detection sensibility, and meanwhile there are problems such as perturbation and eddy occurring.
(2) Sample Injection Volume
As for the measurement of micro-scale sample injection volume, the most common method is to measure the mass and then convert the result using the density. However, the resolution of a mass measurement method may range from only a mg to μg grade at most, so it is not applicable for measuring the volume of a sample below a μg grade. In addition, in the conventional art, fluorescent materials or dyes are added into a fluid and then the fluorescent absorbance of the fluid is measured and converted into fluid volume. Although significant micro-scale volume may be measured in this way, such as nL, fluorescent materials and dyes have different chemical properties in different fluid media. Therefore, there may be uncertainty and deviation of measurement due to drift of absorption wavelength or quench of absorption strength.
Currently, there is no method or technology for measuring micro-volume and micro-flow rate by radiochemical substances and radiochemical analytical apparatus in any country in the world. However, the method disclosed in the invention for measurement and calibration of radiochemical substances may effectively address the above-mentioned problems. It not only may adjust the range of measurement and calibration from μL to pL or from μL/min to pL/min, but also is applicable for measurement and calibration of sample injection volume, spray volume, dipper adhesive volume and delivery rate of mobile phase. Thus, the invention can effectively address difficulties of prior arts and is expected to have significant influences on the development of the biomedicine, clinical diagnosis, drug screening, nano science and technology and analytical technique in every country in the future.